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© Waters Corp. 2004©2004 Waters Corporation

Troubleshooting Common MS Problems

by Claude Mallet, Ph.D

[email protected]

presented by

Michael S. Young, Ph.D.

©2004 Waters Corporation©2004 Waters Corporation

Troubleshooting Common MS Problems

Overview of Troubleshooting Strategy

ESI sources parameters

Single and triple Quadrupoles

SIR vs MRM

Ion Suppression

Outline


Try to simplify --assess impact on lab efficiency --

inspect the MS or /MS/MS --try to categorize

troubleshoot the easiest to fix items first

CHEMISTRY MECHANICAL IMPROPER SETTINGS

Adducts (Na+, K+)Multiple chargeIon stability (pH)Ion suppression

Ion beam instabilityProbe cloggingHeater/sensorN2 gas flow Loss of vacuumPower supply

ESI sources parametersQuadrupoles parametersAcquisition modes

MS Troubleshooting Strategy


Sample Preparation Chromatography

MassSpectrometry

Polarity:Silica- C18, C8, C4, C2Hybrid- C18, C8, C4, C2Polymer- C18, C8, C4, C2Embedded polar groupCyano, PhenylParticle size:2.5, 3.5, 5 or 7 µmInternal diameter:4.6, 3.9, 2.1, 1.0, 0.32 mm and 75 µmLength:150, 100, 50, 30, 20 mm

Source:ESIAPcINano-ESIMass analyzers:magnetic sectorselectric sectorstime of flightquadrupoleion trapFT-ICR

Raw sample:- CaCO2, microsomes, P450,hepatocytes … etc- tissue, CSF, plasma, serumurine, tears … etc- water, sediment, food … etc

Extracted sampleFor LC/MS/MS

The Total Analysis


BA MassSpectrometry

ESI source parameters

Part 1


Quattro UltimaZQ

Quattro Premier

Mass Spectrometers


First, let’s take a look at the first event in an ESI orthogonal source. The primary function of the probe is to transform a liquid (from LC column or other source) into a gas stream asshown in the red circle. Three parameters are used to optimize the probe, which are the 1- nebulizer gas, 2- the desolvation gas flow and 3-the desolvation temperature. The nebulizer gas is automatically set at maximum on the ZQ and manually on other mass spectrometers (i.e. Ultima, QToF, LCT … etc). The desolvation gas flow and desolvation temperature can be optimized to maximizesignal intensity. Highertemperatures are required when using mobile phasescontaining high percentage of water.

2,31

Mass Spectrometers


Nebulizer gas flow off Nebulizer gas flow on

Notice the formation of a liquid drop. It can lead to source flooding if unattended for a long period of time. To avoid potential electrical hazard, the source is equipped with a drain valve.

Notice the formation of liquid droplets from condensation of the sprayer on the probe holder assembly.

ESI Probe Parameters


Isolation valve

Cone shield and cone assembly

Baffle

Stainless steel capillary

Desolvation heater

Ion Block

ESI probe

ESI Source


10 mm5 mm

Cone

Probe



Capillary Tip

Make sure capillary extends approx. 0.5 mm beyond probe tip.

Any corrosion, deposit constriction or other flow restriction will hinder proper nebulization.


Probe too far from the cone?

Probe extends too far past cone?



Probe too close to the cone?



Response ofreserpine shows a good gaussian distribution with baselineresolution of the C13 isotopes

Temperature and gas flow are parameters that affect the desolvation efficiency of the probe. Improper settings can result in loss of signal. These values are optimized according to the column flow rate.

ESI Probe Parameters (tune page)


In this case, a too lowdesolvation temperature resulted in a 50 % reduction in signal intensity.

This effect is compound dependent.

Desolvation Temperature


Similar loss of signal intensity, in this case, it is due to a too lowsetting of the desolvation gas (113 vs 550 L/hr). The gas used for the desolvation is nitrogen

It must be of high purity (99.95%) and oil free. (traps can be used to increase the gas purity if needed).

Make sure delivery pressure is regulated to 100 psi.

Desolvation Gas Flow


Column flow rate Desolvation temp Desolvation gas flowµL/min °C liters/hr

< 10 100 to 120 200 to 25010 to 20 120 to 250 250 to 40020 to 50 250 to 350 250 to 400>50 350 to 400 400 to 750

Higher desolvation temperatures give increased sensitivity. However, increasing the temperature above the range suggested reduces beam stability. Increasing the gas flow rate higher that the quoted values lead to unnecessary high nitrogen consumption. Avoid operating the desolvation heater for long periods of time without proper gas flow.To do so could damage the source.

Suggested Settings


Dewar tanks Nitrogen generator

Both setups are widely used and the choice mostly depends on the consumption of nitrogen per day. Larger laboratories will have a tendency to choose the nitrogen generator for convenience and cost for long term operation.

Dewar Tank vs Nitrogen Generator


At this point let’s take a look at the second event. Once a spray is stable, ions are produced and directed toward the mass analyzer. Five parameters in the orthogonal source are used for this purpose. These parameters are: 1- capillary voltage, 2- cone voltage, 3-extraction voltage and 4- RF lens (transfer optics) 5- Source temperature. A high voltage, in the kV range, is applied to a stainless steel capillary tubing in the probe. This will produce charged droplets. With the assistance of the desolvation gas flow and desolvation temperature, those droplets will in turn produce ions in gas phase next to the cone. The cone voltage attracts positively charged ions from the spray into a reduced pressure chamber (ion block). The extractor and RF lens are used to guide the ion beam into the mass analyzer

1 2

3 4

ESI Probe

ESI Source

ESI Source Parameters


Clean cone and cone shield

Notice the white residue on the cone shield, but the aperture of the cone is still clear. This is an indication that samples injected on this MS were not clean. In both pictures, the baffle shows brown spots, which indicates routine and normal usage. The white residue can result from long exposure to poorly prepared samples or from nonvolatile mobile phase additives.Over time, the aperture of the cone will become clogged, thus reducing signal intensity.

cone

baffle

Brown spot



These are typical starting values to obtain a stable ion beam with flow rate ranging from 0.2 to 0.4 ml/min.

The ion block is heated to avoid any condensation problems.

The source has a maximum setting of 150 °C.



With insufficientcapillary voltage, the signal shows an 80%decrease in signal intensity.

Typical optimum values for most small molecules are between 3.0 and 3.5 kV.

Higher values usually have little effect on signal intensity.

Deviations from experimentally optimized value may indicate problems in the source.

Capillary Voltage


The cone voltage isapplied to a spherical metal plate, the first gate between the sprayer (at atmospheric pressure) and the inside of the mass analyzer (at 10-6

Torr of pressure). The cone creates the first bend of the ion beam in the orthogonal source.

This slide shows that we have optimized the cone voltage at 35 volts and increased our signalintensity.

Cone Voltage


Poor response can occur if cone voltage is set too low. Asufficient voltage isrequired to atract a high population of ions into the ion block.

Once the cone volatage is optimized, loss of sensitivity may result from contamination at the cone.

Cone Voltage


Poor response can also occur if cone voltage is set too high. Too much energy causes a phenomenon known as “In-sourcefragmentation”.

When ions are accelerated from the sprayer to the ion block with very high velocities, collisions among ions can create a high population of daughter ions at the expense of parentions.

In this case, the ion at m/z 609 shows a 90% reduction in signal intensity.

Cone Voltage


The extractor voltage is applied to a second cone shaped metal plate that separatesthe ion block and the mass analyzer. This plate creates a second 90 degree angle in the ion beam, completing the Z spray shape.

An incorrect voltage setting of the extractor resulted in a 70% reduction in signal intensity.

Typical extractor voltage settings range from 1 to 3 volts;higher values will not usually give better sensitivity. Higher than expected values may indicate contamination in the source block

Extractor Voltage


The RF lens focuses the ion beam as it passes into the mass analyzer. In the tandem mass-spectrometer, it focuses the beam to the center of thetransition lens hexapole assembly.

The RF lens valueshould typically be set to range from 0.1 to 0.5 volts.

RF Lens


3.5

3.5

3. 3.

In the example shown, we needed to increase the RF lens to achieve a symmetrical peak shape. This may indicate that the source is contaminated.

RF Lens


BA MassSpectrometry

ESI source parametersQuadrupoles

Part 2


The quadrupole mass analyzer, like other type of mass analyzers (I.e. ToF, ion traps, sector … etc) separates ions according to their mass to charge ratio (m/z). The quadrupole is made of 4 highly polished metal rods positioned at precise angles from one another. These rods are connected to high voltage power supply (DC, positive/negative) and a radio frequency (RF) generator. The slope of RF/DC applied to the rods is proportional to a range or a specific mass to charge ratio.

Quadrupole Mass Analyzers


Source

DetectorNonresonant Ion

Resonant Ion

dc and rf voltages

+Udc + V cosωt

-Udc – V cosωt

Molybdenum Alloy

Quadrupole Schematic


Stable ion

Non-resonantTrajectory

Pre-Filters

ResonantTrajectory

Quadrupole

Resonant vsNon-Resonant Trajectory


Quadrupole Unit Mass Resolution


572.8339

570 571 572 573 574 575 576 577m/z0

100

%

0

100

%

573.9185

574.8116

573.2997

574.3072

575.3155

QuadrupoleResolution: 1000

Q-ToFResolution: 10 000

[M+H]+

Isotopes

Bradykinin Frag 1-5:Arg-Pro-Pro-Gly-Phe


Quadrupole

Resolution: 1000

285 286 287 288 289m/z0

100

%

0

100

%

286.4118

287.1521

287.6505

288.1563

[M+H]+2

Q-ToF

Resolution: 10 000

Isotopes

Bradykinin Frag 1-5:Arg-Pro-Pro-Gly-Phe


The low and high mass resolution are arbitrary values that are calculated from the RF/DC ratio.

The LM setting affects the resolution of ions at the low mass range of the quadrupole; the HMsetting at the high mass range of the quadrupole.

The quadrupole can only achieve mass unit resolution, which means that multiple chargedpeaks are not fully resolved.

Low andHigh Mass Resolution


.

Udc

V

(DC voltage)

(Rf voltage)

Correct V/U ratiomass 1 & 2 are resolved

R: 1000

V/U slope

Stable trajectory

Unstable trajectory

Quadrupole Stability Diagram


If LM and HM resolution are set too low, the quadrupole acts as a transmission cell (RF only). The isotope peaks merge with the main peak.

Notice the increase insignal intensity at the expense of a significant loss of resolving power.

On the other hand, if the values are too high, the quadrupole is over resolved with resulting poor sensititivity.

Low and High Mass Resolution


.

Udc

V

(DC voltage)

(Rf voltage)

Low V/U ratiomass 1 & 2 merge together

R: 10V/U slope

Stable trajectory

Unstable trajectory



In this case, the LH and HM resolution were set too high. The ion beam falls in the nonresonantportion of the stability diagram shown earlier.

Under these conditions, the ion beam will not reach the multiplier at the back of the mass spectrometer and produce a signal.

Low and High Mass Resolution


.

Udc

V

(DC voltage)

(Rf voltage) Incorrect V/U ratiomass 1 & 2 are over resolved

V/U slope

Unstable trajectory

Stable trajectory



The ion energy is applied to a small lens positioned between the quadrupole and the multiplier. This lens is used to refocus the beam toward the multiplier. Typical values range from 0.3 to 0.6.

As shown here, higher values will produce distortion and loss of resolution betweenthe peak and isotopes.

Ion Energy


The multiplier is the last step in the signal production. The ionsproduced by the ESI source and filtered by the quadrupole are converted by the multiplier into ameasurable current.

If the multiplier is set too low, as shown here, the signal intensity will be considerably reduced. Too high a multiplier setting produces saturation (flat-top peaks) and poor quantitation.

Multiplier


BA MassSpectrometry

MS/MS

Part 3


Single Ion Recording(SIR Mode)

Static

Full Scan(MS mode)

ScanningLOQ = 500 pg (quantity injected) LOQ = 5 pg (quantity injected)

Note: A quadrupole mass spectrometer is typically available with amass range of 2000 Daltons or 4000 Daltons

Single Q Mode of Acquisition


Full Scan Acquisition


0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

Scan ES+TIC

2.06e92.76

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

Scan ES+TIC

3.12e92.76

2.56

3.182.98

3.29

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

Scan ES+TIC

5.60e93.18

2.962.54 2.743.27

[ ] = 50 ng/mL

[ ] = 5 ng/mL mixture of 5 basic compounds

[ ] = 500 ng/mL

Full Scan Acquisition


Single Ion Recording (SIR)


0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

SIR of 5 Channels ES+TIC

3.77e72.76

2.55

0.85 2.26

3.182.97

3.28

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

Scan ES+TIC

2.06e9

[ ] = 5 ng/mL

[ ] = 5 ng/mL

Scan mode

SIR mode

Single Ion Recording (SIR)


A single quadrupole mass analyzer can be operated in two distinct modes, SCAN and SIR. A triple quadrupole mass spectrometer can offer 4 types of acquisition; 1- Daughter scan, 2- Multiple Reaction Monitoring (MRM), 3- Parent scan and 4- Constant neutral loss or gain scan. These types of scans rely on the middle quadrupole called the “collision cell”. The collision cell is in fact a hexapole (6 rods) that operates in RF mode only (no resolution capacity). The cell can be pressurize with argon gas. This provides a physical surface onto which ions filtered by MS1 can be fragmented by collision, hence the term “collision induced dissociation”. Depending if MS1 and MS2 are set in scan or park mode will determine the desired type of acquisition mentioned earlier.

Tandem Mass SpectrometryCollision Induced Dissociation (CID)


Parent Ion Scanning

MS1 MS2Collision

Cell

StaticScanning

Triple Q Modes of Acquisition


Daughter Ion Scanning

MS1 MS2Collision

Cell

Static Scanning



Constant Neutral Loss or Gain

MS1 MS2Collision

Cell

ScanningScanning



Multiple Reaction Monitoring

MS1 MS2Collision

Cell

Static Static



MS1 MS2Daughter

MS1

CID

MS2

A triple quadrupole mass spectrometer offers lowersensitivity and reproducable fragmentation. With Multiple Reaction Monitoring (MRM), up to 1000x in sensitivity can be achieved in comparison to scan mode. The next slides will describe some of the common problems associated with MRM and a guide on how to optimize MRM transitions.

We infused a basic drug (clemastine) and openedwindows for MS1, daughterand MS2. Notice the mass unit resolution of the parent mass and isotopes on both MS1 and MS2.

Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively

Optimizing an MRM


Next, the LM/HM (1)values are lowered to the point that the first isotope and the parent ion are both passed into the collision cell. The peaks in the MS1 windows (2) will broaden and show loss of resolution. Conequently,the ion beam passing from MS1 to the collision cell is also increased (3). In the daughter scan window (middle window in the tune page), the parent peak is offscale and one isotope of the molecule is evident.Since MS2 is set with unit mass resolution setting (LM/HM = 15 ), good resolution is seen in the third window among the parent peak and the isotopes.

1

23


Optimizing an MRM


1

2

In this slide, LM/HM on MS1is slightly increased ( small gain in resolution) just to the point that the isotope is not seen. This step is crucial, if LM/HM on MS1 are too low, additional ions will enter the collision cell and will create additional daughter ions for each isotope of the parent molecule.


Optimizing an MRM


1 2

3

Then, by decreasing the LM/HM on MS2 (1), the signal in the daughter scan window has increased (3). The resolution on MS2 also decreases as a consequence of lowering the LM/HM values (2).


Optimizing an MRM


Let’s take a look at a common problem when optimizing an MRM transition. If we look at the LM/HM values (1,2) on both MS1 and MS2, the quadrupoles are set at unit mass resolution. This can be verified in the MS1 and MS2window in the tune page. The peaks shows a gaussian distribution and resolution with the isotopes. However, the daughter window in the tune page shows no signal (4). The answer is quite simple; choosing a correct MRM transition also requires us to park MS1 on the top of the parent peak. The parent peak has a molecularweight of 344.2 Da (see previous slide). In this example, the setting was incorrect, 343.7 Da. The difference of 0.5 Da (3) was enough to miss the parent peak completely in MS1, thus leading to a total loss of signal in MS2.

1

2

3

4


Optimizing an MRM


1

2

34

5

At this point, the quadrupolesare optimized to give maximum signal intensity (1,2) and MS1 is correctly set at 344,2 Daltons (4). In this tune page both MS1and MS2 windows were removed so we can concentrate on the daughter ion scan (5). As we can see, the tune page only shows the parent ion without any daughter ions. This is because the collision gas was not activated (6) and the collision cell was not optimized to produce daughters ions from collision with argon gas (3).

6


Optimizing an MRM


Prior to introduction of collision cell gas (1). thepressure on the collision cell pirani guageindicates 1.0 e-4 mbar(2). Also, since there is no argon gas in the collision cell, the analyzer penning guage shouldshow a pressure in the vicinity of 1-2 e-5 mbar (3). This pressure indicates that the entire mass analyzer is under optimum vacuum.

1 2 3

Optimizing an MRM


1

2

When the collision gas button is activated (1),argon gas will flow freely into the collision cell located inside the mass analyzer (between MS1 and MS2). Notice that the pressure on the collision cell gage will increase (2), typical values are between 2 to 3e-4 mbar.

Optimizing an MRMTune Page with Gas Cell Pressure


1 2

3

Once the argon gas pressure is optimized in the collision cell, it requires some energy to produce fragments. In this case, the collision energy is set at 15 (1) (arbitrary units). The result is the production of two major fragments at 215 Da and 128 Da(2) of the parent ion of mass 344.20 Da. Notice that the energy level is still low enough to see a small fraction of the parent ion (3).


Optimizing an MRMSetting Collison Cell Energy


1

23

In this scenario, the collision energy was purposely increased to higher values (1)that gives a 100 % conversion of the parent ion (3) into fragments ions. However, the level of energy is also high enough to produce further fragmentation of smaller daughter ions (2) and to reduce the intensity to the larger fragments. This type of setting is not favored for trace analysis. The optimum for sensitivity is to use conditions that will produce a 100 % conversion of the parent ion into one or two majors fragments. Hhowever, the production of more than two fragments may be desirable for verification of unknowns.


Optimizing an MRMSetting Collison Cell Energy


100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z0

100

%

0

100

%

0

100

%

344.2

215128

215

128344.2

215

128

CID 0 volts

CID 10 volts

CID 20 volts

[M+H]+

NCH3

O

CH3

Cl215

128

Clemastine

(Different scale)

Daughter Ion Spectrum


Note: typical value of dwell times are between 0.2 and 0.05 seconds

Multiple MRM


0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

MRM of 5 Channels ES+TIC

2.91e52.95

2.542.76

3.183.27

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

SIR of 5 Channels ES+TIC

2.29e6

2.27 2.75

2.56

3.182.96 3.27

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0

100

%

Scan ES+TIC

2.04e9

[ ] = 0.1 ng/mLScan mode

[ ] = 0.1 ng/mLSIR mode

[ ] = 0.1 ng/mLMRM mode

Multiple MRM


BA MassSpectrometry

Ion suppression

Causes of Ion SuppressionTroubleshooting Ion Supression

Part 4


What is ion suppression or enhancement ?

* All spectrums at same scale

Ion suppression or enhancement is a known phenomenon that occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.

Scan ES+

Scan ES+

100

0

%

Scan ES+472.63 50/50 Water/ACN + 0.5 % NH 4OH

Peak intensity: 5 143 003 136Signal increase: + 41 %

100

0

%

472.63

50/50 Water/ACNPeak intensity: 3 636 985 856

100

464 466 468 470 472 474 476 478 480 482 484m/z0

%

472.63

50/50 Water/ACN + 0.5 % TFAPeak intensity: 893 059 072Signal decrease: -75 %

Terfenadine* All spectrums at same scale

Ion suppression or enhancement

occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.

Scan ES+

Scan ES+

100

0

%



100

0

%

472.63


100

464 466 468 470 472 474 476 478 480 482 484m/z0

%

472.63


Terfenadine

Ion suppression or enhancement

occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.

Scan ES+

Scan ES+

100

0

%



100

0

%



100

0

%

472.63


100

0

%

472.63


100

464 466 468 470 472 474 476 478 480 482 484m/z0

%

472.63


Terfenadine

100

464 466 468 470 472 474 476 478 480 482 484m/z0

%

472.63


Terfenadine


Various type of additives can increase or decrease the signal of a target analyte. Furthermore, since ESI is compound dependent, it is expected to see variation in signal intensity as well as suppression or enhancement effect. At this point, let’s take a look at common additives used in LC and the response profile of various SPE extraction protocols.

Acidic additive Buffers SPE extracts

Trifluoroacetic acid Ammonium formate protein precipitationAcetic acid Ammonium bicarbonate Oasis HLB 1-DFormic Acid Ammonium biphosphate Oasis HLB 2-D

Oasis MCXBasic additive Ion pairing additive

Ammonium hydroxide Tetraethylammonium hydroxidePyrrolidine Dimethylhexylamine

Detergents

Triton X100SDS

What is ion suppression or enhancement ?


2795 ESI-MS

Infusion pumpUsed to add range of modifiers, salts, ion pairs, pH additives,Matrix extracts

50/50 ACN/ H2O8 compounds

0.2mL/min

0.2mL/min

ES+260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate*411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine

*quaternary amine drug

Compare 50/50 ACN/ H2Oto additive stream signal(triplicates) blank, matrix, blank

Experimental design aimed to look for a better solution –removal of suppression


250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0

100

%

0

100

%

Scan ES+6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60485.6

713.74757.83

801.86

845.88889.91

933.93 977.95

Scan ES+ 7.38e9354.4

260.2

291.3

609.6

485.6472.6

411.4591.6

260.2

260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %

50/50 water/ACN Blank

0.5 % Triton X 100

260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate *411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine* Quaternary amine molecule

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0

100

%

0

100

%

Scan ES+6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60485.6

713.74757.83

801.86

845.88889.91

933.93 977.95

Scan ES+ 7.38e9354.4

260.2

291.3

609.6

485.6472.6

411.4591.6

260.2

260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %


0.5 % Triton X 100


250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0

100

%

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0

100

%

0

100

%

Scan ES+6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60485.6

713.74757.83

801.86

845.88889.91

933.93 977.950

100

%

Scan ES+6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60485.6

713.74757.83

801.86

845.88889.91

933.93 977.95

Scan ES+ 7.38e9354.4

260.2

291.3

609.6

485.6472.6

411.4591.6

354.4

260.2

291.3

609.6

485.6472.6

411.4591.6

260.2

260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %


0.5 % Triton X 100


Surfactant


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

609.6354.4

260.3

291.3

485.6472.6

411.5591.7

Scan ES+ 354.3

260.3291.2

609.6

485.6472.6

411.4591.7

50/50 Water/ACN + 0.5 % FA

50/50 Water/ACN

260.3 + 5 %291.3 - 5 %354.4 - 5 %411.5 - 54 %472.6 - 7 %485.6 - 2 %591.7 - 52 %609.6 + 17 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

609.6354.4

260.3

291.3

485.6472.6

411.5591.7

Scan ES+ 354.3

260.3291.2

609.6

485.6472.6

411.4591.7

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

609.6354.4

260.3

291.3

485.6472.6

411.5591.7

Scan ES+ 354.3

260.3291.2

609.6

485.6472.6

411.4591.7

50/50 Water/ACN + 0.5 % FA

50/50 Water/ACN

260.3 + 5 %291.3 - 5 %354.4 - 5 %411.5 - 54 %472.6 - 7 %485.6 - 2 %591.7 - 52 %609.6 + 17 %

Acidic Additive


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+ 354.5

260.3291.4

471.6

411.5

609.6

485.6 591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.6591.7

260.3 + 10 %294.4 + 4 %354.4 0 %411.5 + 16 %471.6 + 57 %485.6 + 46 %594.7 + 37 %609.6 - 6 %

50/50 Water/ACN + 0.5 % NH4OH

50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+ 354.5

260.3291.4

471.6

411.5

609.6

485.6 591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.6591.7

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+ 354.5

260.3291.4

471.6

411.5

609.6

485.6 591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.6591.7

260.3 + 10 %294.4 + 4 %354.4 0 %411.5 + 16 %471.6 + 57 %485.6 + 46 %594.7 + 37 %609.6 - 6 %

50/50 Water/ACN + 0.5 % NH4OH

50/50 Water/ACN

Basic Additive


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 1.95e8

354.44

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 7.38e9354.44

260.28

291.31

609.55

485.62472.57

411.49591.66

591.66

50/50 Water/ACN Blank

50 mM Tetraethylammonium hydroxide260.2 - 100 %291.3 - 100 %354.4 - 88 %411.4 - 100 %472.5 - 100 %485.5 - 100 %591.6 - 94 %609.5 - 100 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 1.95e8

354.44

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 7.38e9354.44

260.28

291.31

609.55

485.62472.57

411.49591.66

591.66

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 1.95e8

Scan ES+ 1.95e8

354.44

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0

100

%

Scan ES+ 7.38e9354.44

260.28

291.31

609.55

485.62472.57

411.49591.66

591.66

50/50 Water/ACN Blank

50 mM Tetraethylammonium hydroxide260.2 - 100 %291.3 - 100 %354.4 - 88 %411.4 - 100 %472.5 - 100 %485.5 - 100 %591.6 - 94 %609.5 - 100 %

Ion-Pairing Reagent


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

591.7472.6411.5

485.6 609.6

Scan ES+ 354.4

260.3

291.3

609.6

485.6472.6

411.5591.7

50/50 Water/ACN + 0.1M NaCl

50/50 Water/ACN

260.3 - 93 %291.3 - 95 %354.4 - 37 %411.5 - 62 %472.6 - 71 %485.6 - 84 %591.7 - 45 %609.6 - 95 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

591.7472.6411.5

485.6 609.6

Scan ES+ 354.4

260.3

291.3

609.6

485.6472.6

411.5591.7

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

591.7472.6411.5

485.6 609.6

Scan ES+ 354.4

260.3

291.3

609.6

485.6472.6

411.5591.7

50/50 Water/ACN + 0.1M NaCl

50/50 Water/ACN

260.3 - 93 %291.3 - 95 %354.4 - 37 %411.5 - 62 %472.6 - 71 %485.6 - 84 %591.7 - 45 %609.6 - 95 %

Salt Adducts


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.6472.6 546.6 609.6

Scan ES+ 354.4

260.2

291.2

609.6

485.6472.6

411.4 591.6

.

50/50 Water/ACN

260.3 - 98 %291.3 - 98 %354.4 - 87 %411.4 - 94 %472.6 - 92 %485.6 - 95 %591.7 - 42 %609.6 - 94 %

50/50 Water/ACN + rat plasma supernatant

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.6472.6 546.6 609.6

Scan ES+ 354.4

260.2

291.2

609.6

485.6472.6

411.4 591.6

.

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.6472.6 546.6 609.6

Scan ES+ 354.4

260.2

291.2

609.6

485.6472.6

411.4 591.6

.

50/50 Water/ACN

260.3 - 98 %291.3 - 98 %354.4 - 87 %411.4 - 94 %472.6 - 92 %485.6 - 95 %591.7 - 42 %609.6 - 94 %

50/50 Water/ACN + rat plasma supernatant

Rat Plasma


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6

50/50 Water/ACN + human plasma supernatant

50/50 Water/ACN

260.2 - 97 %291.2 - 96 %354.4 - 86 %411.4 - 93 %472.6 - 93 %485.6 - 95 %591.6 - 89 %609.5 - 93 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260.2 - 97 %291.2 - 96 %354.4 - 86 %411.4 - 93 %472.6 - 93 %485.6 - 95 %591.6 - 89 %609.5 - 93 %

Human Plasma


Condition/Equilibrate1.0 mL methanol / 1.0 mL water

Load1.0 mL plasma

Wash1.0 mL 5% methanol in water

Elute0.5 mL MeOH

Dilute with 0.5 ml water

Plasma Sample

* 30 mg HLB 96 plate

Reversed Phase SPE


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

260.1 291.2

591.6

472.5

411.4485.5

609.5

Scan ES+ 354.2

260.2

291.2

609.5

485.4472.5

411.4591.6

50/50 Water/ACN + rat plasma HLB 1D extract

50/50 Water/ACN

260.2 - 41 %291.2 - 26 %354.4 - 9 %411.4 - 32 %472.6 - 23 %485.6 - 38 %591.6 + 26 %609.5 - 49 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

260.1 291.2

591.6

472.5

411.4485.5

609.5

Scan ES+ 354.2

260.2

291.2

609.5

485.4472.5

411.4591.6


50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

354.4

260.1 291.2

591.6

472.5

411.4485.5

609.5

Scan ES+ 354.2

260.2

291.2

609.5

485.4472.5

411.4591.6


50/50 Water/ACN

260.2 - 41 %291.2 - 26 %354.4 - 9 %411.4 - 32 %472.6 - 23 %485.6 - 38 %591.6 + 26 %609.5 - 49 %

Reversed Phase SPE - Rat Plasma


Condition/Equilibrate1.0 mL methanol / 1.0 mL water

Load1.0 mL plasma

Prepare Sample Solution

Wash 21.0 mL MeOH

Elute0.5 mL MeOH + 2% NH4OH

Dilute with 0.5 ml water

Wash 11.0 mL Water + 2 % FA

Locks basic drugon ion exchanger

Removes polar interferences

* 30 mg Oasis MCX 96 well plate

Mixed Mode Cation-Exchange SPE


260.2 - 9 %291.2 - 11%354.4 - 0.5 %411.4 - 13 %472.6 - 9 %485.6 - 2 %591.6 - 8 %609.5 - 8 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

609.6

472.6

411.5

485.6

591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.5 591.7

50/50 Water/ACN + rat plasma MCX extract

50/50 Water/ACN

260.2 - 9 %291.2 - 11%354.4 - 0.5 %411.4 - 13 %472.6 - 9 %485.6 - 2 %591.6 - 8 %609.5 - 8 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

609.6

472.6

411.5

485.6

591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.5 591.7


50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0

100

%

0

100

%

Scan ES+

354.4

291.3260.3

609.6

472.6

411.5

485.6

591.7

Scan ES+ 354.4

260.3291.3

609.6

485.6472.6

411.5 591.7


50/50 Water/ACN

Mixed Mode SPE – Rat Plasma

Troubleshooting Common MS Problems - Vijaya …vijayaanalyticals.com/pdf/waters LCMS Troubleshooting.pdf · Troubleshooting Common MS Problems ... ESI sources parameters ... stable,

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